Probing the translational dynamics of influenza virus infection.

At the cellular level, the outcome of virus infection hinges on a battle between virus and cell for the control of the cellular biosynthetic machinery, as this ultimately determines the amount of progeny virus produced. Many viruses encode their own nucleic acid polymerases, but all are reliant on the cellular protein synthesis machinery. The ribosome therefore represents the key battleground. Using the major human and animal pathogen influenza A virus (IAV) as a model system, we will use the novel technique of ribosomal profiling to detail this conflict at a level of detail hitherto impossible. Ribosomal profiling allows the accurate determination of which mRNAs (viral or cellular) that the ribosomes are translating at a given moment; thereby defining the state of the 'frontline' in the battle for control of the cell. Our objectives are to:

1. Define the cellular and viral mRNAs that are translated in virus-host cell combinations where either the virus 'wins' or where the host wins.

2. To define which of the many viral mechanisms that affect cellular gene expression are and their specific targets which decide the outcome of the battle for control of the cellular translation machinery.

3. Test the hypothesis that IAV specifically manipulates the ribosome to bias it towards preferential translation of viral messengers.


Overall, the project will define the key events from both virus and cellular perspective that define whether an individual infected cell is productively infected; data that will aid the design of new therapeutic intervention strategies.

In order to produce progeny virions, the major human and animal pathogen influenza A virus (IAV) uses the host cell translational machinery to synthesize viral proteins. Simultaneously, the virus attempts to inhibit cellular macromolecular synthesis; both to free up the translation machinery and to block the synthesis of antiviral response proteins. IAV has evolved several seemingly redundant shutoff mechanisms, highlighting the importance of this aspect of virus infection. However, whilst this reinforces this subject as an area for potential therapeutic intervention, the key molecular events from either virus or host perspective are obscure.

Our objectives are to:

1. Utilise the recently developed technique of ribosomal profiling in conjunction with RNAseq to define the cellular and viral 'translatome' in permissive (293T and A549 cells) and non-permissive (monocyte-derived macrophages) cell types and with human-adapted (A/PR/8/34, A/Udorn/72) and avian (A/mallard/NL/10/99) strains of virus.

2. To define the key viral mechanisms and cellular targets that decide the outcome of the battle for control of the cellular translation machinery, by comparing the translatomes of wild type and mutant strains of IAV disabled for specific shut-off functions; PA-X mutants, PB2/PA mutants defective in RNA Pol II degradation and various NS1 mutants defective for CPSF, PAF1, eIF4GI binding.

3. Test the hypothesis that IAV specifically manipulates the biogenesis and/or composition of ribosomes to bias them towards viral translation, using an unbiased affinity purification method followed by mass spectrometry as well as testing specific hypotheses involving viral interactions with the NOH61 helicase and the PELP1, LAS1L, TEX10 and WDR18 complex.


The project will define the key events from both virus and cellular perspective that define the output of progeny virions from an individual infected cell; data that will aid of new therapeutic intervention strategies.

1. A deeper understanding of influenza virus.
IAV is the etiologic agent of influenza, one of the most economically significant diseases of man. The virus causes great morbidity, substantial mortality and has the potential to spread in devastating pandemics. Rapid evolution of IAV persistently complicates the effectiveness of vaccines and therapeutics; presently, there is no effective treatment, and repeated vaccination is necessary against newly circulating strains. A better understanding of the complex host cell pathways co-opted by influenza virus for replication, the means by which the virus gains control and (conversely), the ways in which the cell successfully defends itself, will provide new targets and strategies for antiviral therapy. This information will also be of interest to the biomedical industry and to the general public; IAV is an economically attractive target for the former, and an endless source of interest to the latter.

2. Ribosomal profiling - a new tool in the armoury
Presently, our knowledge of how cells respond to infection, and how the virus counteracts this, is incomplete, particularly at the level of protein synthesis. Furthermore, we know little of how IAV interacts with the host translation apparatus, nor how the cell's translatome is modulated by the many host cell shut off strategies the virus can employ. Ribosomal profiling can provide the ability to rapidly identify specific host factors involved in these pathways, allowing for novel disease intervention strategies. It is a relatively new technique but being increasingly used in the study of translation in healthy and disease states. The progress we have made so far in optimising the system - and the insights will undoubtedly gain during the course of this work - together with the bioinformatics pipeline in development with our collaborator Andrew Firth, leads us to believe that our findings will be of broad use to many others in the field of translation and beyond.

3. Ribosomal profiling - insights into gene expression
Based on published work and our own preliminary work, it is highly likely that the ribosomal profiling studies will identify novel ribosomal pausing signals and unusual examples of translational control and even (potentially) novel genes. All of this information will increase our understanding of the diversity of gene expression in mammalian cells and virus-infected cells. This information will be of wide scientific interest and may have implications in biotechnology and disease.

4. Training and career development.
The grant will provide significant benefit to the careers of the junior staff involved. It will enable Drs Wise and Irigoyen to develop their independent careers, as well as training the junior PDRA at the RI in a wide range of scientific and transferable skills that will benefit their future development. We also anticipate that our internal presentations of the research within our host Institutions will provoke interest in a new technology that will lead to our training other researchers in the technique.